Project Summary Type II diabetes mellitus (T2DM) is marked by aberrant distribution of lipid species, such as incompletely oxidized non-esterified fatty acids in skeletal muscle. However, the mechanism by which fatty acids reach the muscle, and in particular how they transverse across the endothelium barrier, is largely unknown. We have uncovered that the poorly-studied metabolite 3-hydroxyisobutyrate (3-HIB) plays a novel role in fatty acid uptake. Produced in the skeletal muscle as a product of valine catabolism, 3-HIB induces trans-endothelial fatty acid transport both in vitro and in vivo. Valine is a branched-chain amino acid (BCAA), a class of molecules recently shown in a number of epidemiological studies to be implicated in insulin resistance. The discovery of 3-HIB?s role in fatty acid uptake opens a new avenue for exploring the connection between BCAA flux, lipid deposition, and insulin resistance. Moreover, fatty acid transport proteins (FATPs) have been shown to play a major role in fatty acid uptake in numerous tissues, and, intriguingly, knockdown of FATP3 and FATP4, the predominant FATPs in endothelial cells, abrogate 3-HIB mediated fatty acid uptake in these cells. Together, these observations lead to us to hypothesize that 3-HIB is a dominant paracrine modulator of trans-vascular FATP3/4- mediated fatty acid transport, and that excess BCAA catabolism and 3-HIB leads to inappropriate lipid accumulation and insulin resistance. We propose to examine the molecular nature of 3-HIB, FATP3 and FATP4, and discern how they work in concert to induce fatty acid transport across the endothelium. These questions will be addressed by various methods: a genome-wide, high-throughput screen to identify critical components of the pathway; cell culture experiments identifying the subcellular localization and the relevance of the acyl-CoA synthase activity of FATP3/4; and studies in newly generated genetic models of endothelial cell specific FATP3/4 knockout mice. Ultimately, we hope to use these investigations to validate these new targets for the prevention of lipid accumulation in non-adipose tissues, and thus for therapeutic intervention for treating patients with T2DM.